The contractile function of the heart is controlled by the electrical activity initiated in the sinoatrial node and propagated throughout the heart. Specific ion channels generate the action potential and are regulated directly by membrane voltage or by ionic blockade. The excitatory inward and inhibitory outward ionic currents associated with the cardiac action potential are directly modulated by membrane voltage while the background potassium current (IK1) rectifies upon membrane depolarization due to intracellular polyamine (e.g. spermine) block of this anomalous inward rectifier potassium channel. Gap junctions are typically open at rest and, hence, do not require voltage dependent activation. {This project has provided new evidence indicating} that the transjunctional voltage-dependent inactivation possesses rapid kinetics (< 20 ms) at peak action potential voltages that can produce 40-60% reductions in junctional conductance during the duration of the action potential plateau. These same investigations reported the reactivation (recovery) {and facilitation} of the inactivated junctional conductance during the repolarization and early diastolic phases of the ventricular action potential. Slowed conduction is a hallmark property of reentrant arrhythmias and triggered activity arising during or after repolarization are likely mechanisms for premature extrasystoles that initiate reentrant tachycardia and eventual fibrillation. We will investigate the regulation of atrial and ventricular gap junctions and specific connexin gap junctions formed by connexins -40, -43, and -45 by pacemaker, atrial, and ventricular action potential waveforms at different rates of stimulation ranging from 240 (tachycardia) to 30 (bradycardia) beats/min. The same polyamines that modulate lK1 were shown by this laboratory to specifically block connexin40, but not connexin43 gap junctions. Thus, we have demonstrated previously unknown regulatory mechanisms for gap junctional communication that are also associated with the modulation of cardiac excitability. We will continue to examine the molecular basis for ionic blockade by intracellular polyamines or tetra-alkylammonium ions, the transjunctional voltage gating properties, and the gap junction channel conductance using site-directed mutagenesis of distinct connexin40 and connexin43 specific sequences. Several connexin amino terminal {and other} mutations already {proposed} will be further examined to {define} the connexin-specific {determinants} of {transjunctional voltage gating}, channel conductance, ion permeation, and occlusion. These unique cytosolic and pore- forming domain sequences represent possible therapeutic targets for regulating cardiovascular gap junctional communication.